Studies on the Reconstitution of O2-Evolution of Chloroplasts 1

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Extraction of spinach (Spinacia oleracea L.) chloroplasts with cholate-asolectin in the absence of Mg2+ results in the rapid and selective inactivation of O2 evolution and a partial (30 to 40%) loss of photosystem II (PSII) donor activity without extraction of thylakoid bound Mn (∼5 to 6 Mn per 400 Chlorophyll). Inclusion of ethylene glycol in the extractions inhibits loss of O2 evolution and results in quantitative and qualitative differences in proteins solubilized but does not significantly inhibit the partial loss of PSII donor activity. Similarly, in two stage experiments (extraction followed by addition of organic solvent and solubilized thylakoid protein), O2 evolution (V and Vmax) of extracted chloroplasts is enhanced approximately 2.5- to 8-fold. However, PSII donor activity remains unaffected. This reversal of cholate inactivation of O2 evolution can be induced by solvents including ethanol, methanol, 2-propanol, and dimethyl sulfoxide. Such enhancements of O2 evolution specifically required cholate-solubilized proteins, which are insensitive to NH2OH and are only moderately heat-labile. NH2OH extraction of chloroplasts prior to cholate-asolectin extraction abolishes reconstitutability of O2 evolution. Thus, the protein(s) affecting reconstitution is unlike those of the O2·Mn enzyme. The specific activity of the protein fraction effecting reconstitution of O2 evolution is greatest in fractions depleted of the reported Mn-containing, 65-kilodalton, and the Fe-heme, 232-kilodalton (58-kilodalton monomer), proteins. Divalent (∼3 millimolar) and monovalent (∼30 millimolar) cations do not affect reconstitution of PSII donor activity but do affect reconstitution of O2 evolution by decreasing the protein(s) concentration required for reconstitution of O2 evolution in nonfractionated, cholate-asolectin extractions. The data indicate a reconstitution of the PSII segment linking the PSII secondary donor(s) to O2-evolving centers.

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